Methods and compositions for modulating an immune response with immunogenic oligonucleotides

ABSTRACT

This document relates to compositions and methods for modulating an immune response. For example, compositions of immunostimulatory CpG oligonucleotides derived from retroviral genomes are provided.

CLAIM OF PRIORITY

This application is a continuation application from U.S. Non-provisionalapplication Ser. No. 13/544,129, filed on Jul. 9, 2012 which is adivisional application from U.S. Non-provisional application Ser. No.12/572,494, filed on Oct. 2, 2009, claims priority to U.S. ProvisionalApplication Ser. No. 61/234,431, filed on Aug. 17, 2009, and U.S.Provisional Application Ser. No. 61/102,641, filed on Oct. 3, 2008,which are incorporated by reference in their entirety herein.

TECHNICAL FIELD

This document relates to compositions and methods for modulating animmune response. For example, this document provides compositions ofimmunostimulatory CpG oligonucleotides derived from retroviral genomes.

SEQUENCE LISTING

A text file named Sequence_Listing.txt created on Jul. 9, 2012 with afile size of 18.9 KB which is incorporated herein by reference as iffully set forth at length is being filed concurrently herewith using theEFS-Web file system.

BACKGROUND

The cells of the innate immune system, such as dendritic cells (DCs),provide an immune response that does not depend upon specific antigenrecognition. See, for example, Tosi, J. Allergy Clin. Immunol.116:241-49 (2005). Dendritic cells lack the highly specific antigenreceptors of T and B cells, and rely instead on a set of patternrecognition receptors (“PRRs”), which recognize and bindpathogen-associated molecular patterns and thereby transduce an immuneresponse signal. Fabrick et al., J. Biol. Chem. 279:26605-11 (2004).Illustrative of the class of PRRs are the Toll-like receptors (“TLR5”),which recognize a range of molecular patterns and generate intracellularsignals for activating a number of host responses. Toll-like receptorssuch as TLR-9 detect microbial DNA by recognizing the presence ofunmethylated cytidine-guanosine (CG) dinucleotides within certain basecontexts, or “CpG motifs.” The interaction between microbial DNA andTLR-9 receptor induces cell signaling pathways which includemitogen-activated protein kinases and NFκB. These signaling events, inturn, provoke leukocyte gene expression and cytokine secretion. CpGmotifs are not prominent in vertebrate genomes due to a phenomenon knownas “CpG suppression,” but are present at the expected frequency inprokaryotic DNA. This contrast has been attributed to evolution of thevertebrate immune system to recognize unmethylated CpG motifs andrespond with a coordinated cytokine response.

SUMMARY

The methods and materials provided herein are based in part on thediscovery that ODNs of desired immunogenicity can be designed on thebasis of CG motifs endogenous to a viral or retroviral genome such asthe Human Immunodeficiency Virus-1 (HIV-1) genome. As described herein,immunogenic oligonucleotides can be isolated nucleic acids containingCpG motifs that have nucleotide sequences derived from the HIV-1 viralgenome, the Simian Immunodeficiency Virus (SIV) genome, or another viralor retroviral genome. In some cases, the isolated nucleic acids can havesequences having modifications or substitutions relative to HIV-1, SIV,or other retroviral genomes. As described herein, such immunogenicoligonucleotides can be used to stimulate lymphocytes to produce animmune response as determined by stimulated cytokine production. In somecases, oligonucleotides can be used to modulate the immune response asdetermined by altered cytokine production. The methods and materialsprovided herein can allow a clinician or other medical professional torestore immune function in a mammal with an immunodeficiency disorderand/or to treat an infection or other disorder by modulating cytokineproduction.

In general, this document features an isolated nucleic acid. Theisolated nucleic acid can consist of from 20-84 contiguous nucleotidesof a mammalian retroviral sequence. The isolated nucleic acid cancomprise at least one CpG motif, with the proviso that at least onenon-thymidine nucleotide 1, 2, or 3 bases immediately 5′ or immediately3′ to the CpG motif is substituted with thymidine. The isolated nucleicacid can be 20-50 nucleotides in length. The isolated nucleic acid canbe 24 nucleotides in length. The isolated nucleic acid can comprise asequence selected from the group consisting of SEQ ID NO:28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84. The mammalianretroviral sequence can be a HIV genomic sequence. The mammalianretroviral sequence can be a SIV genomic sequence.

In another aspect, this document features an isolated nucleic acid. Theisolated nucleic acid can have a nucleotide sequence consisting of asequence selected from the group consisting of SEQ ID NO:28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84.

In another aspect, this document features an isolated nucleic acid. Theisolated nucleic acid can have a nucleotide sequence consisting of SEQID NO:26. The isolated nucleic acid of can comprise one or morephosphate backbone modifications. The isolated nucleic acid can compriseone or more nucleotide analogues. The isolated nucleic acid of any ofclaims 1-7 can comprise at least one stabilizing element.

In another aspect, this document features a composition. The compositioncan comprise the nucleic acid of any of claims 1-10 and a therapeuticantigen.

In another aspect, this document features a method for stimulatinglymphocyte cytokine production. The method can comprise contactinglymphocytes with the nucleic acid of any of claims 1-10 under conditionswherein the cytokine production is enhanced relative to uncontactedlymphocytes. The contacted lymphocytes can form primed lymphocytes. Themethod can further comprise administering the primed lymphocytes to amammal. The contacting can be in vivo. The contacting can furthercomprise application of a therapeutic antigen. The stimulation can bemeasured according to a TLR-9 antagonism assay. The cytokine can beselected from the group consisting of IL-6, IL-10, IL-12, and TNF-α, orany combination thereof. The administering can comprise intranasal,oral, transdermal, intranasal, parenteral, intraperitoneal, intrathecal,rectal, or vaginal administration.

In another aspect, this document features a method for stimulatinglymphocyte cytokine production. The method can comprise contactinglymphocytes with an isolated nucleic acid under conditions wherein saidcytokine production is enhanced relative to uncontacted lymphocytes. Theisolated nucleic acid can consist of a nucleotide sequence selected fromthe group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, and 74.The contacted lymphocytes can form primed lymphocytes. The method canfurther comprise administering the primed lymphocytes to a mammal. Thecontacting can be in vivo. The contacting can further compriseapplication of a therapeutic antigen. The stimulation can be measuredaccording to a TLR-9 antagonism assay. The cytokine can be selected fromthe group consisting of IL-6, IL-10, IL-12, and TNF-α, or anycombination thereof. The administering can comprise intranasal, oral,transdermal, intranasal, parenteral, intraperitoneal, intrathecal,rectal, or vaginal administration.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph exemplifying standard measurement curves (absorbancevs. concentration) for Interleukin-10 (IL-10), with respect to anenzyme-linked immunosorbent assay (ELISA) for monitoring theimmuno-responses of Namalwa cells in vitro.

FIG. 2 is a graph exemplifying standard measurement curves (absorbancevs. concentration) for Interleukin-6 (IL-6), with respect to an ELISAfor monitoring the immune response of Namalwa cells in vitro.

FIG. 3 is a graph exemplifying standard measurement curves (absorbancevs. concentration) for Tumor Necrotic Factor-α (TNF-α), with respect toan ELISA for monitoring the immune response of Namalwa cells in vitro.

FIG. 4 is a graph showing time-course curves for cytokines IL-10, IL-6,and TNF-α according to the immune response elicited by CpG ODN inNamalwa cells.

FIG. 5 is a bar graph depicting the level of IL-10 production by Namalwacells after treatment with control (no ODN), ODN 2006, ODN C274, and ODNT-C274.

FIG. 6 is a bar graph depicting IL-10 and TNF-α production by Namalwacells treated with DNA fragments of different length.

FIG. 7 is a series of bar graphs, each graph depicting the level ofproduction of IL-10 (A), TNF-α (B), and IL-6 (C), respectively, byNamalwa cells stimulated by one of 25 ODNs having a sequence derivedfrom the HIV genome.

FIG. 8 is a series of bar graphs, each graph depicting the level ofproduction of IL-10 (A), TNF-α (B), and IL-6 (C), respectively, byNamalwa cells stimulated by one of 25 ODNs, each ODN a variant of an ODNpresented in FIG. 5.

FIG. 9 is a bar graph depicting IL-10 and IL-6 cytokine production ninedays after treatment with ODNs. C=control (naïve) cells; ED-SL-17,ED-SL-13 and ED-SL-10=“educated” Namalwa cells. C+SL-17, C+SL-13 andC+SL-10=naïve cells treated with respective modified ODNs. C+S17, C+S13,C+S10=naïve cells treated with respective unmodified ODNs.ED-SL-17+SL17, ED-SL-13+SL-13 and ED-SL-10+SL-10=educated cellssubjected to a second treatment with respective modified ODNs.ED-SL-17+S17, ED-SL-13+S13 and ED-SL-10+S10=educated cells subjected toa second treatment with respective unmodified ODNs. Data are presentedas mean±SEM.

FIG. 10 is a bar graph depicting IL-10 and IL-6 cytokine production ninedays after treatment with modified and unmodified HIV-1 ODNs. C=control(naïve) cells; ED-S23 and ED-SL-4=“educated cells” Namalwa cells. C+S23,C+SL-23, C+S4, and C+SL4=naïve Namalwa cells treated with respectiveunmodified and modified ODNs. ED-S23+S23, ED-S23+SL-23, ED-SL-4+S4, andED-SL-4+SL-4=educated Namalwa cells subjected to a second treatment withrespective modified and unmodified ODNs.

FIG. 11 presents a series of bar graphs depicting the production ofcytokines Interleukin-12 (IL-12) (A), TNF-α (B), and IL-6 (C) in monkeylymphocytes following treatment with SIV-derived ODNs. Data arepresented as mean±SEM.

FIG. 12 presents a series of bar graphs depicting the production ofcytokines IL-12 (A), TNF-α (B), and IL-6 (C) in human peripheral bloodmononuclear cells (PBMCs) following treatment with SIV-derived ODNs.Data are presented as mean±SEM.

FIG. 13 presents a series of bar graphs depicting the production ofcytokines IL-12 (A), TNF-α (B), and IL-6 (C) in human PBMCs followingtreatment with HIV-derived ODNs.

FIG. 14 presents a series of bar graphs depicting the production ofcytokines IL-12 (A), TNF-α (B), and IL-6 (C) in human lymphocytesfollowing treatment with 2 sets of HIV-derived ODNs. Data are presentedas mean±SEM.

FIG. 15 presents a series of bar graphs depicting the production ofcytokines IL-12 (A), TNF-α (B), and IL-6 (C) in monkey lymphocytesfollowing treatment with 2 sets of HIV-derived ODNs. Data are presentedas mean±SEM.

FIG. 16 is a bar graph depicting IL-12 and IL-6 cytokine production inhuman PBMCs 5 days after treatment with a HIV-derived ODN. C=control(naïve) human PBMCs. ED-S23=“educated cells” human PBMCs. C+S23=naïvehuman PBMCs treated with the unmodified ODN. ED-S23+S23=educated humanPBMCs subjected to a second treatment with unmodified ODN S23.

FIG. 17 is a bar graph depicting IL-12, TNF-α, and IL-6 cytokineproduction in monkey PBMCs 6 days after treatment with a SIV-derivedODN. C=control (untreated) monkey lymphocytes. C+C-11=control cellstreated with ODN C-11. ED-C-11=educated cells with ODN C-11.ED-C11+C-11=educated cells treated with ODN C-11 six days after thefirst treatment.

DETAILED DESCRIPTION

The methods and materials provided herein are based in part on thediscovery that oligonucleotides (ODNs) of desired immunogenicity can bedesigned on the basis of CG motifs endogenous to a retroviral genomesuch as the Human Immunodeficiency Virus-1 (HIV-1) genome. As describedherein, immunogenic ODNs can be isolated nucleic acids containing CpGmotifs that have nucleotide sequences derived from a retroviral genome(e.g., the HIV-1 viral genome, the SIV genome). In some cases, theisolated nucleic acids can have sequences having modifications orsubstitutions relative to HIV-1, SIV, or other mammalian retroviralgenomes. As described herein, such immunogenic oligonucleotides can beused to stimulate lymphocytes to produce an immune response asdetermined by stimulated cytokine production. In some cases,oligonucleotides potentially can be used to suppress an immune responseas determined by reduced cytokine production.

Immunogenic Nucleic Acids

This document provides methods and materials related to isolated nucleicacid molecules that can be immunogenic oligonucleotides. The term“nucleic acid” as used herein encompasses both RNA and DNA, includingcDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, andnucleic acids having modified backbones, such as phosphorothioatenucleic acids, peptide nucleic acids, and morpholinos. The nucleic acidcan be double-stranded or single-stranded. Where single-stranded, thenucleic acid can be the sense strand or the antisense strand. In somecases, the nucleic acid can be circular or linear. As provided herein,an isolated nucleic acid can be about 8-84 nucleotides in length (e.g.,about 8-15, 8-20, 9-20, 10-20, 11-25, 12-27, 15-30, 16-35, 18-35, 20-40,20-45, 21-46, 22-47, 23-48, 24-49, 25-50, 30-50, 40-60, 45-65, 50-64,50-70, 55-70, 55-75, 60-74, 65-60, 65-74, or 70-84 nucleotides). In somecases, the isolated nucleic acid can be about 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84nucleotides in length.

The term “isolated” as used herein with reference to nucleic acid refersto a naturally-occurring nucleic acid that is not immediately contiguouswith both of the sequences with which it is immediately contiguous (oneon the 5′ end and one on the 3′ end) in the naturally-occurring genomeof the organism from which it is derived. An isolated nucleic acidincludes, without limitation, a recombinant DNA that exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as recombinant DNA that is incorporated into a vector,an autonomously replicating plasmid, a virus (e.g., a retrovirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid can include arecombinant DNA molecule that is part of a hybrid or fusion nucleic acidsequence. It will be apparent to those of skill in the art that anatural nucleic acid existing among hundreds to millions of othernucleic acid molecules within, for example, cDNA or genomic libraries,or gel slices containing a genomic DNA restriction digest, is not to beconsidered an isolated nucleic acid.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence. It will beapparent to those of skill in the art that a non-naturally-occurringnucleic acid existing among hundreds to millions of other nucleic acidmolecules within, for example, cDNA or synthetic nucleic acid librariesis not to be considered an isolated nucleic acid.

The isolated nucleic acids provided herein can have nucleotide sequencesderived from a mammalian retroviral genome. For example, isolatednucleic acids can be synthesized to have sequences derived from theHIV-1 viral genome or from the SIV viral genome. An exemplary retroviralgenome for the methods and materials provided herein can be the9719-nucleotide full-length genome of the HIV-1 HXB2 strain (GenBankAccession No. K03455). The HXB2 strain is the most infectious HIV inNorth America and Europe, and is widely used as a reference strain. See,e.g., Korber et al. (Eds), Human retrovirus and AIDS 1998: A compilationand analysis of nucleic and amino acid sequences, Los Alamos NationalLaboratory, USA (1998). In some cases, a retroviral genome can be thefull-length genome of the Sykes strain of SIV. In some cases, theisolated nucleic acids provided herein can have nucleotide substitutionsor modifications relative to a retroviral genome (e.g., the HIV-1 viralgenome). For example, an isolated nucleic acid can have mononucleotide(e.g., T) or dinucleotide (e.g., TT) substitutions adjacent to a CGmotif or “core.” Isolated nucleic acids can be selected based on thenumber of “CG” cores within a 24-mer sequence and the flanking sequencesurrounding each “CG” core. For example, isolated nucleic acids can have1, 2, 3, 4, 5, or more “CG” cores.

Modifications or substitutions to increase or decrease theimmunostimulatory activity of a nucleic acid having CpG core can includemodification of nucleotides within several nucleotides (e.g., 1, 2, 3,4, or 5 nucleotides) 5′ and within several nucleotides (e.g., 1, 2, 3,4, or 5 nucleotides) 3′ of a CpG core (i.e., those nucleotides “adjacentto” the core). Modifications can be made according to the anticipatedimmunostimulatory effect of individual nucleotides. For example, theranking of individual nucleotides, in order of decreasing impact, can beas follows: T>A>C>G. For RNA, the order can be as follows: U>A>C>G. Inview of this ranking, an isolated nucleic acid with sequence derivedfrom a retroviral genome (e.g., the HIV-1 genome) as described hereincan be modified by, for example, substituting G with T. Such amodification can enhance the stimulatory effect of the modified ODN,relative to its unmodified counterpart, while substituting T with G candecrease the stimulatory effect. Isolated nucleic acids, therefore, canbe selected according to the number of “CG” cores and the flankingsequence surrounding each “CG” core. Exemplary modifications of isolatednucleic acids derived from HIV-1 and SIV retroviral genomes aredescribed in Examples 4 and 5, respectively.

The isolated nucleic acids can be relatively resistant to degradationby, for example, endonucleases and exonucleases. Secondary structures(e.g., stem loops, palindromic sequences) can be used as stabilizingelements to stabilize the nucleic acids against such degradation.Nucleic acid stabilization can be accomplished via phosphate backbonemodifications. To this end, a stabilized ODN can have at least a partialphosphorothioate-modified backbone, which may be significant to theviral entry-blocking activity described herein. See Luganini et al.,Antimicrob Agents Chemother. 52(3):1111-20 (2008). The pharmacokineticsof phosphorothioate ODNs have demonstrated a systemic half-life ofapproximately 48 hours in rodents, generally suggesting the utility ofbackbone-modified ODNs for in vivo applications. See, for example,Agrawal et al., Proc. Nat'l Acad. Sci. USA 88:7595-99 (1991).

Phosphorothioates can be synthesized via automated techniques thatemploy phosphoramidate or H-phosphonate chemistries. Aryl- andalkyl-phosphonates can be synthesized as described, for example, in U.S.Pat. No. 4,469,863. Alkylphosphotriesters, in which the charged oxygenmoiety is alkylated, can be prepared by automated solid-phase synthesisusing commercially available reagents. Any other appropriate method formodifying a nucleic acid backbone and making substitutions can be used.See, for example, modification methods described by Uhlmann and Peyman,Chem. Rev. 90:543-84 (1990); Goodchild, Bioconjugate Chem. 1(3):165-87(1990); and U.S. Pat. Nos. 7,105,495 and 7,176,296.

Any appropriate method including, without limitation, common molecularcloning and chemical nucleic acid synthesis techniques can be used toobtain isolated nucleic acid molecules. For example, isolated nucleicacids can be chemically synthesized, either as a single nucleic acidmolecule or as a series of oligonucleotides. In some cases,oligonucleotides can be synthesized de novo using any of a number ofprocedures widely available in the art. Exemplary methods of synthesiscan include the β-cyanoethyl phosphoramidite method (Beaucage et al.,Tet. Let. 22:1859-1862 (1981)) and nucleoside H-phosphonate method(Garegg et al., Tet. Let. 27:4051-4054 (1986); Froehler et al., Nucl.Acid Res. 14:5399-5407 (1986); Garegg et al., Tet. Let. 27:4055-4058(1986); and Gaffney et al., Tet. Let. 29:2619-2622 (1988)). Thesemethods can be performed by a variety of commercially-availableautomated oligonucleotide synthesizers. Alternatively, recombinantnucleic acid techniques, including restriction enzyme digestion andligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA),can also be used to isolate a nucleic acid molecule provided herein. Insome cases, isolated nucleic acids can be obtained using the polymerasechain reaction (PCR). PCR refers to a procedure or technique in whichtarget nucleic acid is amplified in a manner similar to that describedin U.S. Pat. No. 4,683,195, and subsequent modifications of theprocedure described therein. General PCR techniques are described, forexample, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequenceinformation from the ends of the region of interest or beyond are usedto design oligonucleotide primers that are identical or similar insequence to opposite strands of a potential template to be amplified.Any appropriate oligonucleotide primer can be used.

In some cases, the immunogenic oligonucleotides provided herein can havedetectable labels or probes attached. Such detectable labels can permitdetection in the presence of a complementary sequence based on detectionof a bound label. Administration and detection of detectable immunogenicoligonucleotides may be useful for diagnosing certain diseases that arecaused or exacerbated by particular nucleic acid sequences (e.g.,systemic lupus erythematosus, sepsis, autoimmune diseases).

Methods of Using Immunogenic Oligonucleotides

This document also provides methods and materials for stimulating animmune response. In some cases, the isolated nucleic acids providedherein can be used to induce or amplify an innate immune response. Forexample, induction or amplification of an immune response results fromThI-type activation. Accordingly, the isolated nucleic acids can serveas oligonucleotide immunogens and can be administered to a mammal (e.g.,a human subject) in an amount effective for inducing or amplifying suchan immune response. Other exemplary mammals for the methods andmaterials provided herein can include, without limitation, non-humanprimates, horses, cows, goats, dogs, cats, rabbits, rats, and mice.

In some cases, isolated nucleic acids can be used to stimulatelymphocyte cytokine production. For example, a method for stimulatinglymphocyte cytokine production can include contacting lymphocytes withan isolated nucleic acid provided herein. Isolated nucleic acidsappropriate for such a method can have the exact sequences providedherein. In some cases, isolated nucleic acids appropriate for such amethod can have any additional sequence, e.g., at least one additionalCpG core sequence and/or a stabilizing sequence. For example, additionalnucleotide sequence, e.g., a retroviral sequence having at least one CpGcore, can be added to either terminus of an isolated nucleic acidprovided herein. Stimulation of lymphocyte cytokine production accordingto the methods provided herein can be in vivo or in vitro.

In some cases, the isolated nucleic acids can be administered to amammal in order to address an immune system deficiency. For example, theoligonucleotide immunogens provided herein can be used to enhance orrestore innate and antigen-specific acquired immune responses in mammalswith impaired immune systems, such as those infected by HIV-1. Enhancingor restoring an immune response according to the methods provided hereincan provide a mode of eradicating latent HIV in infected mammals treatedwith antiviral therapy. In some cases, the methods can be used tosuppress viral transmission in mammals considered to be at high risk forbecoming infected.

Isolated nucleic acids provided herein can be used as oligonucleotideimmunogens and administered to a mammal for therapeutic or prophylacticpurposes. For example, the oligonucleotide immunogens can beadministered to a mammal in order to treat or prevent a viral infection,retroviral infection, bacterial infection, or parasitic infection. Animmunogenic oligonucleotide can be used to block virus entry, therebyaiding in the elimination of the virus. In some cases, isolated nucleicacids can be administered as a therapeutic immunogen (e.g., a vaccineadjuvant) for the prevention or treatment of an allergy or foranti-cancer therapy. In some cases, a method of treating or preventingcan include administering an isolated nucleic acid provided herein to amammal having or at risk of having such an infection in an amounteffective for treating or preventing infection of the mammal. Infectionssuitable for the methods and materials provided herein can includeinfection by hepatitis virus, HIV, hepatitis B, hepatitis C, herpesvirus, papilloma virus, intracellular bacteria (e.g., Mycobacteriumtuberculosis), and intracellular parasites, in particular non-helminthicparasites. Other exemplary medical conditions for which the isolatednucleic acids can be used can include microbial infections (e.g.,sexually transmitted disease), fungal diseases (e.g., candidiasis), orparasitic diseases including, without limitation, malaria, pneumocystiscamii pneumonia, leishmaniasis, cryptosporidiosis, and toxoplasmosis.

In some cases, the isolated nucleic acids potentially can be used todecrease a mammal's immune response. For example, isolated nucleic acidscan have modifications that suppress CpG-mediated cytokine induction ina mammal. Such isolated nucleic acids likely can be used forimmunomodulation in a mammal having an autoimmune disease or symptomsassociated with enhanced cytokine production. In some cases, suchisolated nucleic acids can be administered to a mammal in order todecrease or suppress overstimulation of an immune response such as inthe case of an autoimmune disease, allergic reaction, asthma, oranaphalaxis. Exemplary autoimmune diseases appropriate for the methodsand materials provided herein can include, without limitation, lupuserythematosus, Addison's disease, alopecia, Guillain Barré syndrome,celiac disease, Crohn's disease, and multiple sclerosis.

For administration in vivo, immunogenic oligonucleotides can beassociated with a molecule that results in higher affinity binding totarget cell (e.g., B-cell, NK cell) surfaces and/or increased cellularuptake by target cells to form an “oligonucleotide delivery complex.”The immunogenic oligonucleotides provided herein can be ionically orcovalently associated with appropriate molecules using techniques thatare well known in the art. A variety of coupling or crosslinking agentscan be used (e.g., protein A, carbodiimide,N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP)). The immunogenicoligonucleotides alternatively can be encapsulated in liposomes orvirosomes using well-known techniques. For anti-cancer therapy,immunogenic oligonucleotides can be administered to a mammal prior tochemotherapy in order to increase responsiveness of the malignant cellsto subsequent chemotherapy and/or to induce natural killer (NK) cellactivity.

Evaluating Immunomodulatory Activity

The immunogenic oligonucleotides can be used to stimulate lymphocytes toproduce an immune response as determined by stimulated cytokineproduction. In some cases, the oligonucleotides can be used to suppresslymphocytes to decrease or suppress an immune response as determined byreduced cytokine production. The activity of the isolated nucleic acidsfor modulation of the immune response (e.g., lymphocyte stimulatoryactivity) can be evaluated by any appropriate methods. For example,immunomodulatory activity can be measured in assays evaluating cytokineproduction/release, including those described in the Examples below. Insome cases, peripheral blood mononuclear cells (PBMCs) can be used toevaluate stimulation of an immune response. In some cases,immunomodulatory capacity of the isolated nucleic acids can be evaluatedusing a TLR-9 antagonism assay. In such an assay, the Namalwa cell line,a human lymphoblastoid cell line derived from a child with a Burkitt'slymphoma, can be used to evaluate stimulation of cytokine (e.g., IL-6,IL-10, and TNF-α) production. Generally, immunosuppressiveoligonucleotides (TLR-9 antagonists) can inhibit TLR-9 agonist-inducedcytokine production in a dose-dependent manner. By contrast,immunostimulatory oligonucleotides (TRL-9 agonists) can stimulate immuneactivation in Namalwa cells as determined by stimulation of inflammatorycytokine production. Direct and indirect CpG ODN-induced cytokinesecretion occurs in a variety of cell types, including B lymphocytes,monocytes, macrophages, dendritic cells, natural killer cells, and evenmast cells. Depending on the cell type activated, they proliferate,upregulate MHC class I and II, B7-1 and B7-2 co-stimulatory molecules,or express a broad range of cytokines including IL-1, IL-6, IL-10,IL-12, IFN-α, IFN-γ, and TNF-α.

In some cases, immunomodulation can be evaluated by detectinginflammatory cytokine release by dendritic cells, lymphocytes,macrophages, T cells, or other immune system cells. Secretion ofcytokines such as IL-12, IL-6, IL-1b, IL-10, and TNF-α, can be measured,for example, by an enzyme linked immunosorbent assay (ELISA).

Activation of naïve T-cells can be assayed by, for example, measuringthe incorporation of ³H-thymidine into newly synthesized DNA inproliferating cells, by measuring induction of cytolytic T-cellactivity, or by detecting T-cell activation markers (e.g., CD44 orCD69). In some cases, expression or translocation of NF-κB can bemeasured by, for example, cell staining with an antibody against NF-κB.Antibodies against NF-κB are available from, for example, Cell SignalingTechnologies, Inc. (Beverly, Mass.).

Any appropriate method can be used to evaluate an individual's immuneresponse following administration of an adjuvant containing at least oneisolated nucleic acid as described herein. For example, a biologicalsample from an individual can be examined to evaluate the immuneresponse in vitro. The biological sample can be blood (e.g., whole bloodor serum), blood cells (e.g., lymphocytes, monocytes, eosinophils, orbasophils), or a mucosal sample (e.g., saliva or gastric andbronchoalveolar lavages). In some cases, biological samples arecollected prior to and after administration of a nucleic acid asdescribed herein.

Preparing Immunogenic Oligonucleotide Compositions

This document also provides methods and materials for obtainingcompositions containing isolated nucleic acids (e.g., an isolatednucleic acid of 8-84 nucleotides derived from a retroviral sequence).For example, isolated nucleic acids can be combined with a therapeuticantigen (e.g., an immunogenic peptide) to form a composition foradministration to a mammal. Suitable therapeutic antigens include, forexample, polypeptides or fragments of polypeptides expressed by tumorsand pathogenic organisms. Examples of therapeutic antigens include,without limitation, propionyl CoA carboxylase (NM_(—)000282), dystrophin(M92650), p53 (M14695), factor IX (BC109215), herpes virus thymidinekinase (NC 001798), measles H and F fusogenic glycoproteins (DQ227321),sodium iodide symporter (NM_(—)000453), and heat shock protein (L12723).Thus, the isolated nucleic acids can be used to generateimmunostimulatory compositions. Such a composition generally contains atleast one isolated nucleic acid as described herein. A composition canbe used as an adjuvant to stimulate a mammal's immune response,typically against an antigen. Compositions can be administered by anyroute that permits uptake of the oligonucleotides by the appropriatetarget cells. Exemplary routes of administration can includeadministration to mucosal surfaces. Mucosal surfaces include, forexample, intranasal, oral, parenteral, rectal, and vaginal surfaces and,accordingly, compositions can be administered by a route includingintranasally, orally, transdermally, gastrointestinally, rectally,vaginally, or via the genitourinary tract. In some cases, immunogenicoligonucleotides can be administered by injection (e.g., subcutaneous,intravenous, parenteral, intraperitoneal, intrathecal). Administrationby injection can involve a bolus or continuous infusion of acomposition.

In some cases, the isolated nucleic acids can be formulated intopharmaceutical compositions using techniques and procedures well knownin the art (see, e.g., Ansel, Introduction to Pharmaceutical Dose Forms,Fourth Edition 1985, 126). For example, a composition can beadministered with a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and anti-fungalagents, isotonic and absorption delaying agents, and the like,compatible with the intended route of administration. For example,pharmaceutical preparations such as sterile solutions or suspensions canbe used to make compositions suitable for parental administration.Pharmaceutically acceptable carriers can also include a solid,semi-solid, or liquid material that acts as a vehicle, carrier, ormedium for the isolated nucleic acids provided herein. The use of suchpharmaceutically acceptable carriers with compositions is well known inthe art. Except insofar as any conventional pharmaceutically acceptablecarrier is incompatible with the active compound, use thereof in any ofthe compositions described herein is contemplated.

Compositions can contain therapeutically effective amounts of one ormore of the isolated nucleic acids and a pharmaceutically acceptablecarrier. Typically, an adjuvant is administered to a mammal such thatthe mammal produces a greater immune response toward an antigen comparedto the immune response produced toward the antigen in the absence of theadjuvant. A suitable amount of an adjuvant is one that increases animmune response in a mammal but does not result in significant toxicity.A suitable amount of an adjuvant can depend on factors such as, withoutlimitation, the route of administration; the nature of the composition;the weight of the mammal; the particular antigen; and the concurrentadministration of other vaccines or drugs. A suitable amount of anadjuvant can be established by one of ordinary skill in the art throughroutine trials establishing dose response curves. For example, atherapeutically effective concentration may be determined empirically bytesting the isolated nucleic acids in in vitro and in vivo systems, andthen extrapolating therefrom for doses for humans or other subjects.

For therapeutic indications, immunostimulatory doses can range, forexample, from about 1 μg to about 10 mg and, more typically, can rangefrom about 10 μg to 1 mg oligonucleotides per administration of theactive agent. Doses can be administered, for example, daily or weekly.Doses for parenteral delivery of a therapeutic composition can range,for example, from about 0.1 μg to about 10 mg oligonucleotides peradministration which could be given daily, weekly, monthly, or accordingto any other appropriate dosing schedule. More typically, parenteraldoses can range, for example, from about 10 μg to about 5 mgoligonucleotides per administration, and most typically from about 100μg to 1 mg oligonucleotides, with about 2-4 administrations being spaceddays or weeks apart.

In some cases, a composition of isolated nucleic acids as describedherein can be administered with one or more additional components. Forexample, the composition can be administered with a penetration enhancerto promote the efficient delivery to a mucosal surface of an adjuvantprovided herein. For example, a penetration enhancer can be a surfactant(e.g., sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether); a fatty acid (e.g., oleic acid, lauricacid, myristic acid, palmitic acid, and stearic acid); a bile salt(e.g., cholic acid, dehydrocholic acid, and deoxycholic acid); achelating agent (e.g., disodium ethylenediamine tetraacetate, citricacid, and salicylates); or a non-chelating non-surfactant (e.g.,unsaturated cyclic urea).

In some cases, methods for solubilizing components of a pharmaceuticalcomposition can be used. Such methods are known to those of skill inthis art, and include, but are not limited to, using cosolvents, such asdimethylsulfoxide (DMSO), polyethylene glycol (PEG) (e.g., PEG400),cyclodextrins or cremaphor; using surfactants, such as TRITON® X-100detergent (Union Carbide Corp.) or TWEEN® polysorbate surfactant (CrodaInternational PLC), or dissolution in aqueous sodium bicarbonate.

Pharmaceutical compositions can be provided for administration to humansand animals in unit dose forms, such as sterile parenteral solutions orsuspensions, containing suitable quantities of the nucleic acidsprovided herein. In some cases, the compositions can be formulated andadministered in unit-dose forms or multiple-dose forms. The term“unit-dose form” refers to physically discrete units suitable for humanand animal subjects and packaged individually as is known in the art.Each unit-dose form contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredimmunomodulatory effect, in association with any necessarypharmaceutical carrier, vehicle or diluent. Examples of unit-dose formsinclude ampoules and syringes and individually packaged tablets orcapsules. Unit-dose forms may be administered in fractions or multiplesthereof. A multiple-dose form is a plurality of identical unit-doseforms packaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, and the like, to thereby form a solution orsuspension. If the composition is to be administered intravenously,suitable carriers can include physiological saline or phosphate bufferedsaline (PBS), and solutions containing thickening and solubilizingagents, such as glucose, polyethylene glycol, and polypropylene glycoland mixtures thereof. In some cases, the composition to be administeredcan also include non-toxic auxiliary substances such as wetting agents,emulsifying agents, solubilizing agents, pH buffering agents and thelike, for example, acetate, sodium citrate, cyclodextrin derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents.

Any appropriate method of preparing such dose forms can be used. Forexample, dose forms can be prepared according to the methods provided inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 15th Edition, 1975.

Articles of Manufacture

This document also provides methods and materials for providing theimmunogenic oligonucleotides or pharmaceutical compositions describedherein as articles of manufacture (e.g., kits) containing packagingmaterial, an immunogenic oligonucleotide within the packaging material,and a label that indicates that the immunogenic oligonucleotide orcomposition is useful for stimulating an immune response, or for thetreatment or prevention of viral infections, bacterial infections, orallergic response.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907; 5,052,558; and 5,033,252. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, pumps, bags, vials, containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Synthesis of HIV-Derived ODNs

Analysis of the 9719-nucleotide HXB2 genome revealed 94 CpG dinucleotidemotifs or “cores” distributed throughout the genome between positions 24to 9650. The cores were numbered according to the scheme described inKorber et al., available athiv.lanl.gov/content/sequence/HIV/REVIEWS/HXB2.html on the World WideWeb. In view of this distribution, twenty-five ODNs were constructedusing conventional synthesis methodology described previously. See, forexample, Stein et al., Nucleic Acids Res. 16:3209-3221 (1988). The ODNswere designed according to CpG-containing portions of the genome of theHXB2 strain of HIV-1. Each of the resultant ODNs was 24 nucleotideslong. Sequences of the 25 HIV-derived ODNs (S1-S25) are presented inTable 1. The corresponding portions collectively spanned the viralgenome from position 24 to position 9472, and, respectively, extendedinto the following HIV-1 genes: gag (p17, portion 13; p24, portion 14;p7, portion 15), pol (p51, portion 16; p31, portion 17), vif (portion18), vpu (portion 19), and env (gp120, portions 20, 21, and 22; gp41,portions 23 and 24). Portions 1-13 and 25 also extended into with 5′ LTRand 3′ LTR, respectively.

TABLE 1 Sequences of HIV-Derived CpG Oligonucleotides Name Core PositionSequences S1 BTI-HXB2-CG24 TTCACTCCCAACGAAGACAAGATA (SEQ ID NO: 1) S2BTI-HXB2-CG238 AATGGATGACCCGGAGAGAGAAGT (SEQ ID NO: 2) S3 BTI-HXB2-CG273GGTTTGACAGCCGCCTAGCATTTC (SEQ ID NO: 3) S4 BTI-HXB2-CG336ACTGCTGACATCGAGCTTGCTACA (SEQ ID NO: 4) S5 BTI-HXB2-CG359AAGGGACTTTCCGCTGGGGACTTT (SEQ ID NO: 5) S6 BTI-HXB2-CG408CTGGGGAGTGGCGAGCCCTCAGAT (SEQ ID NO: 6) S7 BTI-HXB2-CG565TAGTGTGTGCCCGTCTGTTGTGTG (SEQ ID NO: 7) S8 BTI-HXB2-CG639TCTAGCAGTGGCGCCCGAACAGGG (SEQ ID NO: 8) S9 BTI-HXB2-CG687AGGAGCTCTCTCGACGCAGGACTC (SEQ ID NO: 9) S10 BTI-HXB2-CG728ACGGCAAGAGGCGAGGGGCGGCGA (SEQ ID NO: 10) S11 BTI-HXB2-CG751ACTGGTGAGTACGCCAAAAATTTT (SEQ ID NO: 11) S12 BTI-HXB2-CG832GAGAATTAGATCGATGGGAAAAAA (SEQ ID NO: 12) S13 BTI-HXB2-CG921CTAGAACGATTCGCAGTTAATCCT (SEQ ID NO: 13) S14 BTI-HXB2-CG1766GGTCCAAAATGCGAACCCAGATTG (SEQ ID NO: 14) S15 BTI-HXB2-CG2274CTCTTTGGCAACGACCCCTCGTCA (SEQ ID NO: 15) S16 BTI-HXB2-CG2840ATACCACATCCCGCAGGGTTAAAA (SEQ ID NO: 16) S17 BTI-HXB2-CG4603CACCGGTGCTACGGTTAGGGCCGC (SEQ ID NO: 17) S18 BTI-HXB2-CG5540GCCTAGTGTTACGAAACTGACAGA (SEQ ID NO: 18) S19 BTI-HXB2-CG6063AGTACATGTAACGCAACCTATACC (SEQ ID NO: 19) S20 BTI-HXB2-CG6656AATAGTAGTAGCGGGAGAATGATA (SEQ ID NO: 20) S21 BTI-HXB2-CG7146GAAAAAGAATCCGTATCCAGAGAG (SEQ ID NO: 21) S22 BTI-HXB2-CG7342AGAAATTGTAACGCACAGTTTTAA (SEQ ID NO: 22) S23 BTI-HXB2-CG8371TTCACCATTATCGTTTCAGACCCA (SEQ ID NO: 23) S24 BTI-HXB2-CG8414CCCGACAGGCCCGAAGGAATAGAA (SEQ ID NO: 24) S25 BTI-HXB2-CG9421ACTGCTGACATCGAGCTTGCTACA (SEQ ID NO: 25)

Example 2 Namalwa Cellular Assay

Namalwa cells, which tend to exhibit strong immunoresponses (e.g.,cytokine release) following ODN treatment, were obtained from theAmerican Type Culture Collection (Manassas, Va.). The Namalwa cell linewas maintained in RPMI medium supplemented with 10% fetal bovine serum.Cells were maintained in a humidified incubator at 37° C. with 90%relative humidity and 5% CO₂.

Prior to each experiment, cells were seeded at a concentration of 1×10⁶cells/mL in 24-well cell culture plates. The cells were treated withdifferent concentrations of one of the 25 ODNs (0.25 μM to 0.5 μM).Supernatants were collected after 8-hour to 48-hour incubations with theODN and analyzed for Interleukin-10 (IL-10), Interleukin-6 (IL-6), andTumor Necrosis Factor-α (TNF-α) production, respectively, using an ELISAMAX™ detection kit (BioLegend, San Diego, Calif.).

With cytokine values thus determined, standard curves for each of IL-10,IL-6, and TNF-α measurement were established as shown in FIGS. 1, 2, and3, respectively. The ability to detect all three cytokinessimultaneously was also confirmed, as demonstrated in FIG. 4. Forexample, the peak concentration of TNF-α was detected following 8 hoursof ODN incubation. IL-6 concentration peaked following 24 hours of ODNincubation, while IL-10 peaked following 32 hours of ODN incubation.

As a preliminary experiment, Namalwa cells were assayed for stimulationof IL-10 production following treatment with control ODNs and an ODNhaving thymidine substitutions. The cells were treated with thefollowing ODNs as described above: ODN CpG-2006(5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:27)); ODN C274(5′-TCGTCGAACGTTCGAGATGAT-3′ (SEQ ID NO:100)); and ODN T-C274(5′-TCGTGCAAGCTTGCAGATGAT-3′ (SEQ ID NO:101)). As demonstrated in FIG.5, thymidine substitutions in ODN C274 dramatically enhanced IL-10production, which is equal to or higher than IL-10 production followingtreatment with ODN 2006. It was also determined that the optimalconcentration for T-C274 was 0.6 μM.

Example 3 Determination of Preferred ODN Length

To determine the preferred DNA fragment length for cytokine stimulationwith HIV derived oligonucleotides, two groups of DNA sequences weredesigned, EX1 and EX2, based on the HIV genome (Table 2). ODNs having24, 44, 64, and 84 nucleotides were tested (Table 2). EX1-24 ishomologous to ODN 2006 but does not contain a CG core. EX1-44, EX1-64,and EX1-84 are different extensions of EX1-24. Each fragment contained44, 64, and 84 bases, respectively. EX2-24 was a modified sequence ofEX1-24 containing four CG cores. EX2-44, EX2-64, and EX2-84 weredifferent extensions of EX2-24. Namalwa cells were treated with each ofthe ODNs at a concentration of 0.5 μM, and cultured supernatants werecollected for measurement of TNF-α and IL-10 by ELISA. As seen in FIG.6, EX1 fragments with short length (24 and 44 bases) did not stimulateIL-10 and TNF-α production. Among the EX2 fragments, EX2-24 was the moststimulatory. Interestingly, no significant difference in stimulation wasobserved between cells treated with EX1-84 or EX2-84. These resultsindicated that the preferred ODN length for stimulating IL-10 and TNF-αcytokine release was 24 nucleotides. No direct correlation was observedbetween immunostimulation and ODN sequence length greater than 44nucleotides, suggesting that specificity declines for ODNs longer than44 nucleotides.

TABLE 2 Selected DNA fragments from the HIV genome Fragment Length NameSequences (nt) EX1-24 TTATTGTTTTATTATTTCCAAATT 24 (SEQ ID NO: 92) EX1-44TGCTTAAAGATTATTGTTTTATTATTTCCAAA 44 TTGTTCTCTTAA (SEQ ID NO: 93) EX1-64TCCTGAGGATTGCTTAAAGATTATTGTTTTAT 64 TATTTCCAAATTGTTCTCTTAATTTGCTAGCT(SEQ ID NO: 94) EX1-84 CTGGGTCCCCTCCTGAGGATTGCTTAAAGATT 84ATTGTTTTATTATTTCCAAATTGTTCTCTTAA TTTGCTAGCTATCT (SEQ ID NO: 95) EX2-24TCGTCGTTTTATCGTTTCCACGTT 24 (SEQ ID NO: 96) EX2-44TGCTTAAAGATCGTCGTTTTATCGTTTCCACG 44 TTGTTCTCTTAA (SEQ ID NO: 97) EX2-64TCCTGAGGATTGCTTAAAGATCGTCGTTTTAT 64 CGTTTCCACGTTGTTCTCTTAATTTGCTAGCT(SEQ ID NO: 98) EX2-84 CTGGGTCCCCTCCTGAGGATTGCTTAAAGATC 84GTCGTTTTATCGTTTCCACGTTGTTCTCTTAA TTTGCTAGCTATCT (SEQ ID NO: 99)

Example 4 HIV-Derived ODNs

To test the immunostimulatory capacity of each HIV-derived ODN, Namalwacells were seeded at a concentration of 1×10⁶ cells/mL in 24-well cellculture plates. Cells were incubated in the presence of HIV-derived ODNs(see Table 1), each ODN with a 24-nucleotide sequence derived fromgenomic DNA between position 24 and position 9472 of the HIV-1 genome.Culture supernatants were collected after 8 hours of incubation forELISA measurements of TNF-α, and after 24 hours for measurement of IL-10and IL-6. The concentration of each ODN was 0.5 μM. As demonstrated inFIG. 7, the HIV-derived ODNs compared favorably to ODN T2006, having thebacterially-derived sequence 5′-TCGTCGTTTTTTCGTTTTTTCGTT-3′ (SEQ IDNO:26), where residues modified relative to ODN CpG-2006(5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:27)) are bolded andunderscored.

Namalwa cells produced the greatest amount of IL-10 in the presence ofODN T2006. Of the HIV-derived ODNs, only S23 and S13 showed robust IL-10production (FIG. 7A). The other HIV-derived ODNs had no effect on IL-10production. Namalwa cells produced the greatest amount of TNF-α in thepresence of ODN T2006 and HIV-derived ODN S23 (FIG. 7B). All otherHIV-derived ODNs had no effect on TNF-α production. It was observed thatNamalwa cells incubated with the HIV-derived ODNs produced comparableamounts of IL-6 as the control ODN (FIG. 7C). S17 and S23 showed minimalstimulation of IL-6 production. All other HIV-derived ODNs wereineffective. Overall, S23 was revealed to be the most potent cytokinestimulator of all of the HIV-derived ODNs as it demonstrated robuststimulation of all three cytokines. These results demonstrate thatHIV-derived ODNs having CpG motifs can be used to stimulate an immuneresponse in vitro.

Example 5 ODNs with Modified HIV Sequence

Twenty-five ODNs having a 24-nucleotide sequence modified relative tothe ODNs derived from HIV-1 sequence were constructed using conventionalsynthesis methodology described previously. Each sequence was modifiedby T or A nucleotide substitution. Table 3 presents the sequences of the25 modified ODNs (SL-1-SL-25) with the positions of all nucleotidesubstitutions identified (bolded and underscored). To assay forimmunostimulatory activity of the modified ODNs, Namalwa cells wereseeded at a concentration of 1×10⁶ cells/mL in 24-well cell cultureplates, and were incubated in the presence of the modified ODNs(SL-1-SL-25). Supernatant collection and ELISA cytokine measurementswere conducted as described above.

As demonstrated in FIG. 8A, almost all HIV-modified ODNs enhanced IL-10production relative to unmodified fragments. HIV-modified ODNs SL-10,SL-13, SL-17, and SL-24 produced the greatest amount of IL-10 relativeto ODN T2006 (FIG. 8A). HIV-modified ODNs SL-10, SL-13, and SL-17produced the greatest amount of TNF-α relative to ODN T2006 (FIG. 8B).Only SL17 stimulated more TNF-α production than T2006. HIV-modified ODNsSL-3, SL-4, SL-7, SL-10, SL-13, SL-17, SL-22, and SL-24 were the beststimulators of IL-6 production (FIG. 8C). Overall, ODNs SL-4, SL-10,SL-13, SL-17, and SL-24 were the most stimulatory for all threecytokines. These results demonstrate that HIV-modified ODNs having CpGmotifs can be used to stimulate an immune response in vitro.

TABLE 3 Sequences of CpG Oligonucleotides with Modified HIV SequenceName Nucleotide Sequence SL-1 TTCACTCCC TT CG TT GACAAGATA (SEQ ID NO: 28) SL-2 AATGGATGA TT CG TT GAGAGAAGT  (SEQ ID NO: 29) SL-3GGTTTGACA TT CG TT TAGCATTTC  (SEQ ID NO: 30) SL-4 ACTGCTGAC T TCG TTCTTGCTACA  (SEQ ID NO: 31) SL-5 AAGGGACTTT T CG T TGGGGACTTT (SEQ ID NO: 32) SL-6 CTGGGGAGT TT CG TT CCCTCAGAT  (SEQ ID NO: 33) SL-7TAGTGTGTG TT CGT T TGTTGTGTG  (SEQ ID NO: 34) SL-8 TCTAGCAGT TT CG TTCGAACAGGG  (SEQ ID NO: 35) SL-9 AGGAGCTCT T TCG T CG T AGGATTC (SEQ ID NO: 36) SL-10 ACG TT AAGA TT CG TT G TT CG T CG T  (SEQ ID NO: 37) SL-11 TT TGGTGAGT T CG TT AAAAATTTT  (SEQ ID NO: 38)SL-12 GAGAATTAGATCG T TGGGAAAAAA  (SEQ ID NO: 39) SL-13 CTAG TT CG TTTCG TT GTTAATCCT  (SEQ ID NO: 40) SL-14 GGTCCAAAAT T CG TT CCCAGATTG (SEQ ID NO: 41) SL-15 CTCTTTGGC TT CG TT CC T TCGT T A  (SEQ ID NO: 42)SL-16 ATACCACAT TT CG T AGGGTTAAAA  (SEQ ID NO: 43) SL-17 CA T CG T TGCTT CG T TTAGG TT CG T   (SEQ ID NO: 44) SL-18 GCCTAGTGTT T CG TTACTGACAGA  (SEQ ID NO: 45) SL-19 AGTACATGT TT CG TT ACCTATACC (SEQ ID NO: 46) SL-20 AATAGTAGT TT CG TT AGAATGATA  (SEQ ID NO: 47)SL-21 GAAAAAGAAT T CGT T TCCAGAGAG  (SEQ ID NO: 48) SL-22 AGAAATTGTA TCG TT CAGTTTTAA  (SEQ ID NO: 49) SL-23* TTCACCATTATCGTTTCAGACCCA(SEQ ID NO: 50) SL-24 TT CG TT AGG TT CG TT GGAATAGAA (SEQ ID NO: 51)SL-25 ACTGCTGAC T TCG TT CTTGCTACA (SEQ ID NO: 52) *SL-23 is unmodifiedrelative to HIV-1 derived SEQ ID NO: 23 (SL-23 = S-23)

Example 6 Education of Namalwa Cells

Naïve Namalwa cells were treated with 0.3 μM ODN CpG-2006 (SEQ ID NO:27)or 0.3 μM of an ODN 2006 variant (listed in Table 5) and allowed toincubate for 48 hours, after which IL-10 production was quantified byELISA. As presented in Table 4, naïve cells demonstrated enhanced IL-10production (320 pg/mL), relative to untreated cells, 48 hours aftertreatment with ODN 2006, while naïve cells treated with the ODN 2006variants exhibited variable IL-10 production. It was observed that,relative to the stimulation of IL-10 following ODN 2006 treatment, noneof the ODN 2006 variants activated significant IL-10 production in naïveNamalwa cells.

TABLE 4 IL-10 Production in Naïve and Educated Namalwa cells FollowingIncubation with ODN 2006 Variants IL-10 Production* Namalwa ODN ODN ODNODN ODN ODN ODN ODN Cells 2006 2006-1 2006-2 2006-3 2006-4 2006-5 2006-62006-7 Naïve 320 230 80 290 120 150 140 260 Educated‡ 440 390 140 450180 340 300 440 *IL-10 production (pg/mL) 48 hours after treatment withODN 2006 and its variants (ODN 2006-1-ODN 2006-7). ‡Educated cells aredefined as cells treated with ODN 2006 (0.3 μM) and allowed to dividefor two weeks.

TABLE 5 Nucleotide Sequences of ODN 2006 Variants ODN 2006T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 27)Changed area

*C*G*

*C*G*N*N*N*N*N*N*C*G*N*N*N*N*N*N*C*G*N*N ODN 2006-1

*C*G*

*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 85) Changed areaN*C*G*N*C*G*N*N*N*N*N*

*C*G*N*N*N*N*N*

*C*G*N*N ODN 2006-2 T*C*G*T*C*G*T*T*T*T*G*

*C*G*T*T*T*T*G*

*C*G*T*T (SEQ ID NO: 86) Changed area N*C*G*N*C*G*N*N*N*N*N*N*C*G*

*N*N*N*N*N*C*G*

*N ODN 2006-3 T*C*G*T*C*G*T*T*T*T*G*T*C*G*

*T*T*T*G*T*C*G*

*T (SEQ ID NO: 87) Changed area N*C*G*N*C*G*N*N*N*N*N*

*C*G*N*N*N*N*N*

*C*G*N*N ODN 2006-4 T*C*G*T*C*G*T*T*T*T*G*

*C*G*T*T*T*T*G*

*C*G*T*T (SEQ ID NO: 88) Changed area N*C*G*N*C*G*N*N*N*N*N*N*C*G*

*N*N*N*N*N*C*G*

*N ODN 2006-5 T*C*G*T*C*G*T*T*T*T*G*T*C*G*

*T*T*T*G*T*C*G*

*T (SEQ ID NO: 89) Changed area N*C*G*N*C*G*N*N*N*N*

*N*C*G*N*N*N*N*

*N*C*G*N*N ODN 2006-6 T*C*G*T*C*G*T*T*T*T*

*A*C*G*T*T*T*T*

*A*C*G*T*T (SEQ ID NO: 90) Changed area N*C*G*N*C*G*N*N*N*N*N*N*C*G*N*

*N*N*N*N*C*G*N*

ODN 2006-7 T*C*G*T*C*G*T*T*T*T*G*T*C*G*A*

*T*T*G*T*C*G*A*

 (SEQ ID NO: 91)

To test the effect of “priming” cells with an immunostimulatoryoligonucleotide, cells treated with ODN 2006 were allowed to incubatefor an additional two weeks during which time they divided normally. Atthe end of two weeks, the treated cells were re-introduced to ODN 2006and introduced for the first time to seven ODN 2006 variants (Table 5),all at a concentration of 0.3 μM. The Namalwa cells appeared to beprimed to the entry of ODN 2006 because IL-10 production was enhancedappreciably in all cases (Table 4). Indeed, the cells became “educated”to recognize the sequence character of ODN 2006 and were “programmed” or“primed” to recognize DNA sequences harboring a similar character (ODN2006-1-ODN 2006-7). In other words, the variant ODNs (Table 5) boostedimmune activity (IL-10 production) in educated Namalwa cells in a mannersimilar to ODN 2006 although they did not activate substantially IL-10production in naïve cells.

To test the response of naïve and educated Namalwa cells to HIV-modifiedODNs, the above-described assays were repeated with the modified CpG ODNT2006 and HIV-modified ODNs (SL-17, SL-13, and SL-10). The treated cellswere named ED-T2006, ED-SL-17, ED-SL-13 and ED-SL-10, respectively. Bothnaïve and treated cells were passaged according to standard cell culturemethods. Nine and 21 days post-treatment, the treated cells weresubjected to a second exposure and incubation with the respective CpGODNs. Stimulation of cytokine (IL-10 and IL-6) production was measuredby ELISA. As demonstrated in FIG. 9, all educated cells maintained anelevated production of IL-10 and IL-6 following the first treatment.Educated cells (ED-SL-17, ED-SL-13 and ED-SL-10) showed increasedproduction of IL-10 and IL-6 after second treatment with thecorresponding modified ODNs at nine days (FIG. 9). After the secondtreatment, all educated cells treated with either modified or unmodifiedHIV-derived ODNs showed cytokine production similar to that of naïvecells exposed to HIV-derived modified ODNs. In sum, these resultssuggest the Namalwa cells became “educated” to recognize the sequencecharacter of HIV-derived ODNs.

To further test the response of Namalwa cells to HIV-derived ODNs, naïveand educated cells were treated with ODNs SL-4 (SEQ ID NO:31), S23 (SEQID NO:23), and SL-23 (SEQ ID NO:50). The ODN SL-4 was chosen because ithas only one CpG island and 87.5% homology with unmodified S4.Unmodified ODN S23 was chosen because it was previously shown tostimulate cytokine production in Namalwa cells. The treated, “educated”cells were named ED-SL-4 and ED-523. Both naïve and educated cells werepassaged in the normal manner. After nine days, the cells were treatedagain with CpG ODNs SL-23 and S23. The production of IL-10 and IL-6cytokines was measured by ELISA. The results are presented in FIG. 10.It was observed that all educated cells maintained an elevatedproduction of IL-10 and IL-6 on day nine following the initial exposureto SL-4 and S23. All educated cells showed increased production of IL-10and IL-6 following a secondary exposure to the corresponding modifiedand unmodified ODNs.

Example 7 SIV-Derived CpG Oligonucleotides

Twenty-two CpG ODNs were synthesized based on the SIV Sykes straingenome. Each SIV-derived ODN was 24 nucleotides in length. Table 6 liststhe unmodified ODN fragments (C-1-C-22) and shows the number andposition of all CpG dinucleotide cores (in bold) and correspondingflanking sequences. Peripheral blood mononuclear cells (PBMCs) wereisolated from monkey (Macaca fascicularis) blood and treated with theunmodified ODNs. Production of IL-12, TNF-α, and IL-6 was measured byELISA as described above. As demonstrated in FIG. 11, all SIV ODNsinduced IL-12 and IL-6 production in monkey lymphocytes. C-8 and C-9induced the highest level of production of all three cytokines andproduction of all three was more robust than with ODN 2006. It wasobserved that not all ODNs stimulated production of TNF-α. For example,ODNs C-3, C-5, C-7, C-17, C-18, and C-20 exhibited no immunostimulatoryeffect on monkey lymphocytes as measured by stimulated TNF-α production.

To test the response of human cells to SIV-derived and HIV-derived ODNs,PBMCs were isolated from a human subject and treated with 22 unmodifiedODNs derived from the SIV genome (C1-C22) and with 25 unmodified ODNsderived from the HIV genome (S1-S25). Production of IL-12, TNF-α, andIL-6 was measured by ELISA. Greater than 50% of the SIV-derived ODNsinduced high-level production of IL-12 (FIG. 12A), TNF-α (FIG. 12B), andIL-6 (FIG. 12C) in human lymphocytes. In some cases, the level ofcytokine production was greater than the cytokine production induced byODN 2006. By comparison, only some HIV-derived ODNs induced cytokineproduction in human lymphocytes (FIG. 13). Overall, the response toHIV-derived ODNs was lower than that induced by ODN 2006 and much lowerthan the response to SIV-derived ODNs.

TABLE 6 Sequences of SIV-Derived CpG Oligonucleotides CG Name SequencesCores C-1 AGGGTGCCATTCGTGCTAGGGTTT (SEQ ID NO: 53) 1 C-2GCCTGGGTGTTCGCTGGTTAGCCT (SEQ ID NO: 54) 1 C-3ACAGAAGCTTTCGAGGCTTGGGAT (SEQ ID NO: 55) 1 C-4CTCGACACGTTCGAGAAGATTAGG (SEQ ID NO: 56) 3 C-5GTCTCCGCGCACGTTAAATGCGTG (SEQ ID NO: 57) 4 C-6GCTTAGGAGTGCGTTATCATGTCT (SEQ ID NO: 58) 1 C-7ATAAACCTGCTCGCTTAGTCGCTA (SEQ ID NO: 59) 2 C-8TTGCTGAGCGTCGGAGAGGGACGA (SEQ ID NO: 60) 3 C-9CGCGACAGGGGCGCGGGTCCCATT (SEQ ID NO: 61) 4 C-10AAGCCTCGACACGTTCGAGAAGAT (SEQ ID NO: 62) 3 C-11CCCGTTGGAACCGACAGGCTCCGA (SEQ ID NO: 63) 3 C-12ATTGTCCGATCCGCTTATCGGGCA (SEQ ID NO: 64) 3 C-13CTCCAGACGGCCGCCGCCTGCAAG (SEQ ID NO: 65) 3 C-14CCCGAAGTTGGCGGTGGAGTACCG (SEQ ID NO: 66) 3 C-15CGGTGGAGTACCGCCCGGACATGT (SEQ ID NO: 67) 3 C-16TGGATCGGAGGCGGTACAGGGGCG (SEQ ID NO: 68) 3 C-17GCTCGCTTAGTCGCTATATTGGAG (SEQ ID NO: 69) 2 C-18TCGCTGGCTTGTAACTCAGTCTCT (SEQ ID NO: 70) 1 C-19CCGAGAGTCTTTGGCTTCTGCTTT (SEQ ID NO: 71) 1 C-20CCGCTAAAATGCTTTAATTGTGGC (SEQ ID NO: 72) 1 C-21CCAATCCGGATTGTAAATTGATTC (SEQ ID NO: 73) 1 C-22ACTGCAATGGGCGCAGCGGCAACA (SEQ ID NO: 74) 2

Example 8 Human and Monkey Lymphocyte Response to ODNs with UnmodifiedHIV Sequence

A second set of CpG ODNs with unmodified HIV sequence were developed totest the cytokine response of human and monkey lymphocytes. Ten ODNshaving a 24-nucleotide sequence unmodified and derived from HIV-1sequence were constructed using the same criteria as reported before forSIV-derived ODNs. Table 7 lists the sequences of the second set ofunmodified HIV-derived ODNs (SA-1-SA-10) and shows the number andposition of all CpG dinucleotide cores (in bold) and correspondingflanking sequences.

To assay the cytokine response of human lymphocytes, peripheral bloodmononuclear cells (PBMCs) were isolated from human blood and treatedwith ODNs SA-1-SA-10 (Table 7) and with eleven previously-testedunmodified ODNs derived from the HIV genome (S1, S4, S5, S9, S12, S13,S17, S18, S23, and S25). Production of cytokines IL-12, TNF-α, and IL-6was measured by ELISA. The results are presented in FIG. 14. It wasobserved that PBMC production of all three cytokines (FIG. 14) washigher in response to the ten new HIV ODNs as compared to thepreviously-tested ODNs.

To assay for the cytokine response of monkey lymphocytes, peripheralblood mononuclear cells (PBMCs) were isolated from monkey blood andtreated with ODNs SA-1-SA-10 (Table 7) and with eleven previously-testedunmodified ODNs derived from the HIV genome (S1, S4, S5, S9, S12, S13,S17, S18, S23, and S25). Production of cytokines IL-12, TNF-α, and IL-6was measured by ELISA. The results are presented in FIG. 15. While PBMCproduction of TNF-α (FIG. 15B) was comparable for both sets of ODNs withunmodified HIV sequences, IL-12 production (FIG. 15A) and IL-6production (FIG. 15C) were pronounced in cells treated with ODNsSA-1-SA-10.

In sum, these data demonstrate that HIV-derived ODNs can exert animmunostimulatory effect as determined by increase cytokine productionin both human and monkey lymphocytes.

TABLE 7 Sequences of HIV-Derived CpG Oligonucleotides CG Name SequencesCores SA-1 GCGCGCACGGCAAGAGGCGAGGGG (SEQ ID NO: 75) 4 SA-2CTCTCTCGACGCAGGACTCGGCTT (SEQ ID NO: 76) 3 SA-3ATCGATGGGAAAAAATTCGGTTAA (SEQ ID NO: 77) 2 SA-4AGAACGATTCGCAGTTAATCCTGG (SEQ ID NO: 78) 2 SA-5GATCCATTCGATTAGTGAACGGAT (SEQ ID NO: 79) 1 SA-6TTCACCATTATCGTTTCAGACCCA (SEQ ID NO: 80) 1 SA-7TAATAATAAGACGTTCAATGGAAC (SEQ ID NO: 81) 1 SA-8TTCGCCACATACCTAGAAGAATAA (SEQ ID NO: 82) 1 SA-9GGAGACAGCGACGAAGAGCTCATC (SEQ ID NO: 83) 2 SA-10GGGACCCGACAGGCCCGAAGGAAT (SEQ ID NO: 84) 2

Example 9 Education of Human Lymphocytes

To test the effect of “priming” human lymphocytes with animmunostimulatory oligonucleotide, peripheral blood mononuclear cellswere isolated from human blood and treated with unmodified ODN S23derived from the HIV genome. After 5 days, educated cells were treatedagain with ODN S23. The production of IL-12 and IL-6 cytokines wasmeasured by ELISA. The results are presented in FIG. 16. The PBMCsappeared to be primed to the entry of ODN S23 because IL-12 productionwas greatly enhanced in educated cells treated with S23 five daysfollowing the first treatment (FIG. 16). Production of cytokine IL-6also was also increased in educated cells in five days after the firsttreatment.

Example 10 Education of Monkey Lymphocytes

To test the effect of “priming” monkey lymphocytes with animmunostimulatory oligonucleotide, peripheral blood mononuclear cellswere isolated from monkey blood and treated with unmodified ODN C-11derived from the SIV genome. Six days after an initial treatment,educated cells were treated again with ODN C-11. The production ofIL-12, TNF-α, and IL-6 cytokines was measured by ELISA. The results arepresented in FIG. 17. The monkey lymphocytes appeared to be primed tothe entry of ODN C-11 because cytokine production was greatly enhancedin educated cells treated with C-11 six days following the firsttreatment (FIG. 17).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for stimulating lymphocyte cytokineproduction comprising contacting lymphocytes with an isolated nucleicacid under conditions wherein said cytokine production is enhancedrelative to uncontacted lymphocytes, wherein said isolated nucleic acidconsists of a nucleotide sequence selected from the group consisting ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, and
 74. 2. The method ofclaim 1, wherein said contacted lymphocytes form primed lymphocytes. 3.The method of claim 2, further comprising administering said primedlymphocytes to a mammal.
 4. The method of claim 1, wherein saidcontacting is in vivo.
 5. The method of claim 1, wherein said contactingfurther comprises application of a therapeutic antigen.
 6. The method ofclaim 1, wherein said stimulation is measured according to a TLR-9antagonism assay.
 7. The method of claim 1, wherein said cytokine isselected from the group consisting of IL-6, IL-10, IL-12, and TNF-α, orany combination thereof.
 8. The method of claim 3, wherein saidadministering comprises intranasal, oral, transdermal, intranasal,parenteral, intraperitoneal, intrathecal, rectal, or vaginaladministration.